Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The invention relates to use of a composition comprising an inhibitor of
adenylyl cyclase in the elongation of circadian rhythm, a method of
extending the period of circadian rhythm in a subject, said method
comprising administering to said subject an inhibitor of adenylyl
cyclase, and to adenylyl cyclase inhibitor for use in the treatment of a
disorder of the circadian rhythm. Preferably the inhibitor is a P-site
inhibitor, preferably 9-(tetrahydrofuryl)-adenine. The composition may
further comprise a JNK inhibitor.

Claims:

1. (canceled)

2. A method of extending the period of circadian rhythm in a subject, said
method comprising administering to said subject a composition comprising
an inhibitor of adenylyl cyclase, wherein said inhibitor of adenylyl
cyclase is a purine site (p-site) inhibitor.

3. A method of modifying the circadian rhythm in a subject comprising
administering to said subject a composition comprising an inhibitor of
adenylyl cyclase, wherein said inhibitor of adenylyl cyclase is a purine
site (p-site) inhibitor.

4. The method of claim 3, wherein said subject is suffering from or at
risk of suffering from jet lag.

6. The method of claim 3, wherein said subject is suffering from or at
risk of suffering from shift lag.

7. The method of any one of claims 2 or 3, wherein said inhibitor of
adenylyl cyclase is a purine site ligand.

8. The method of any one of claims 2 or 3, wherein said inhibitor of
adenylyl cyclase is specific for adenylyl cyclases.

9. The method of any one of claims 2 or 3, wherein said inhibitor of
adenylyl cyclase acts at the P-site of adenylyl cyclase.

10. The method of any one of claims 2 or 3, wherein said inhibitor of
adenylyl cyclase is selected from the group consisting of
9-(tetrahydrofuryl)-adenine, 9-(cyclopentyl) adenine and
2',5'-dideoxyadenosine.

11. The method of any one of claims 2 or 3, wherein said inhibitor of
adenylyl cyclase is 9-(tetrahydrofuryl)-adenine.

12. The method of any one of claims 2 or 3, wherein said composition
further comprises a c-Jun N-terminal kinase (JNK) inhibitor.

26. A pharmaceutical pack or kit according to claim 25 wherein said
adenylyl cyclase inhibitor is 9-(tetrahydrofuryl)-adenine.

27. A pharmaceutical pack or kit according to claim 25 or 26 wherein said
JNK inhibitor is SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one;
1,9-pyrazoloanthrone; SAPK inhibitor II).

Description:

FIELD OF THE INVENTION

[0001]The invention relates to manipulation of the circadian clock. In
particular, the invention relates to slowing or delaying its period in
the treatment of disorders of the circadian clock.

BACKGROUND TO THE INVENTION

[0002]Biological circadian clocks oscillate with an approximately 24-hour
period. They are found in the majority of eukaryotes, as well as many
bacteria. The circadian clock affects many aspects of behaviour and
physiology, causing regular measurable variations in activity over the
24-hour period. The circadian rhythms of sleep, melatonin, secretion and
body core temperature are generated by the suprachiasmatic nucleus of the
hypothalamus, the anatomic locus of the mammalian circadian clock.
Irregularities in the circadian clock underlie a range of clinical
disorders.

[0003]Nearly 20% of employees in industrialized countries are employed, in
shift work, which requires them to drastically change their sleep habits
weekly or even daily. Increasing numbers of employees are needed to work
unconventional shifts. Shift workers having difficulty sleeping during
the day also have difficulty staying awake at work. The more fatigued
they become, the more likely they are to experience "microsleep," an
involuntary bout of sleep brought on by sleep deprivation that lasts for
a few seconds. Approximately 20% of shift workers report falling asleep
during work, which increases the risk of industrial accidents and
decreases productivity.

[0004]There are two main types of shift work. Employees may either work an
unconventional nonfluctuating shift e.g. 11 p.m. to 7 a.m., or rotate
between three different shifts covering the full 24 hour period. Both
types of pattern can cause sleep disorders and related problems affecting
circadian rhythm. Shift work change (SWC, also called `shift lag` or
`shift work sleep disorder`), is caused by a failure to adapt circadian
rhythms to unconventional and/or changing patterns of sleep/wake
behaviour demanded by shift working.

[0005]Treatment for this condition is limited. Behavioral and/or
pharmacological remedies known in the art can only help to alleviate
symptoms. One view in the art is that the body may never fully adapt to
shift work, especially for those who switch to a normal weekend sleep
schedule.

[0006]Delayed sleep phase syndrome (DSPS), also called phase lag syndrome,
is a circadian rhythm sleep disorder. However, unlike jet lag or shift
lag, delayed sleep phase syndrome is a persistent condition. In clinical
settings, it is one of the most common complications of sleep-wake
patterns.

[0007]Delayed sleep phase syndrome results from a desynchronization
between the patient's internal biological clock and the external
environment. In contrast to jet lag, this resynchronization is not
activated by travel or change in external environment. Rather, the
patient's propensity to fall asleep is delayed in relation to that of
normal subjects.

[0008]DSPS patients are typically unable to fall asleep before 2 a.m. and
have extreme difficulty waking early (e.g. by 7 a.m.). The main
difficulty for patients with DSPS is functioning early in the morning. A
Person with DSPS, often fails academically and/or struggles to retain
employment, which can be socially damaging and often compromises their
health in a recent study involving 5,000 participants, DSPS accounted for
about 40% of disorders involving sleep-wake schedules.

[0009]Familial Advanced Sleep Phase Syndrome (FASPS) is characterized by
very early sleep onset and offset. This disorder is especially prevalent
in young adults. Profound phase advance of the sleep-wake, melatonin and
temperature rhythms are characteristic of this disorder. The trait
segregates as an autosomal dominant with high penetrance. There are no
effective treatments for this disorder in the art.

[0010]Jet lag is detrimental to individuals' health and performance. There
are economic problems associated with jet lag such as ineffectiveness
and/or low productivity in the workplace. There are also hazards
presenting an acute risk such as increased chances of accidents due to
fatigue when operating machinery, driving or similar activities.
Currently the only treatment for jet lag is melatonin therapy. This can
produce a minor phase resetting effect on the circadian clock of
approximately 30 minutes/day when administered in the evening. Melatonin
treatment suffers from problems such as having a narrow temporal
therapeutic window, and poor efficacy in humans. Furthermore, melatonin
facilitates sleep and is therefore unsuitable when a period of alertness
or attentiveness is required, for example driving away from an airport on
arrival. Thus, there is a need for improved or alternative therapies to
jet lag and related disorders.

[0011]It should be noted that the circadian clock is so robust that even
temperature perturbations do not affect it. According to ordinary
biophysical predictions, 5° C. reduction in temperature would be
expected to produce a 34 hour clock period. However, despite a slight
dampening the suprachiasmatic nucleus maintains a rhythm of very nearly
24 hours before, during and after treatment. The extreme stability and
resistance of the clock to external factors presents a problem in its
manipulation.

[0012]Fluctuations in cAMP levels have been observed throughout the daily
cycle. cAMP has been regarded as a downstream effector whose levels may
be controlled by the circadian clock. When cAMP has been studied in the
prior art, it has been via acute manipulations. Typically, agonists or
antagonists of the system have been applied and the acute effect
monitored over a maximum of 24 or 48 hours. It has been suggested that
cAMP analogues can produce a phase shift effect in the circadian rhythm.
There has been no suggestion that any effect on the period of oscillation
of the rhythm can be achieved. The view in the art is that cAMP operates
at a level well below that of the circadian clock mechanism.

[0013]Marks and Birabil (2000 Neuroscience Volume 98 pages 311-315)
disclose that infusion of adenylyl cyclase inhibitor into the rat central
nervous system can enhance rapid eye movement sleep. Specifically,
introduction of this compound into particular parts of the rat brain can
produce long lasting increases in the time spent in REM sleep. There is
no mention of circadian rhythm manipulation in this document. It should
be noted that the induction and determination of REM sleep patterns is
accomplished by a mechanism separate from the circadian rhythm. The
frequency and period of REM in sleep is governed by localised brain waves
and electrical pulses during the sleep cycle. Thus, there is no teaching
regarding periodicity or the biological clock in Marks and Birabil.

[0015]cAMP is an important second messenger in all cells. Daily cycles in
cAMP levels can be observed. These cyclical variations in cAMP
concentration were thought to be an output from the circadian clock.
However, it has been surprisingly shown by the inventors for the first
time that cAMP is actually an intrinsic or structural part of the
circadian clock. The invention is based on this remarkable discovery.

[0016]The difference between a structural feature of the circadian clock
and a mere output is profound. There are numerous effectors of the
circadian rhythm, and very many biological events can be seen to
fluctuate or vary regularly throughout the 24 hour day. However, studying
the various outputs or effectors does not permit the manipulation or
adjustment of the fundamental underlying clock mechanism. The
manipulation of the actual biological clock mechanism is rendered
possible for the first time by the present invention. An appreciation of
the significance of this work can be facilitated by comparison to a
mechanical clock. If an operator manipulates the hands of a mechanical
clock, they would appear to change the output. However, it will be
apparent that they have in no way affected the mechanism of the clock,
and the time keeping properties of that clock have not been altered. This
represents the state of the art--at best, crude phase changes or gross
alterations to effectors of the clock can be made. By contrast, according
to the present invention the actual time keeping mechanism, i.e. the
period of the circadian clock can now be manipulated. This is the first
time that this has been possible.

[0017]The present inventors disclose that cAMP is at the heart of the
circadian clock. By manipulating cAMP levels, it is possible to change
the rate at which this clock keeps time i.e. the period of the clock can
be manipulated.

[0018]Thus, in one aspect the invention provides use of a composition
comprising an inhibitor of adenylyl cyclase in the elongation of
circadian rhythm.

[0019]Preferably said inhibitor of adenylyl cyclase is a purine site
(p-site) inhibitor.

[0020]In another aspect, the invention provides a method of extending the
period of circadian rhythm in a subject, said method comprising
administering to said subject a composition comprising an inhibitor of
adenylyl cyclase. The inhibitor is administered in an amount effective to
extend the period of the rhythm by the chosen amount. Guidance regarding
exemplary doses is presented below.

[0021]Elongation has its normal meaning in the art and refers to extension
or prolongation. Preferably elongation means to increase in time, i.e. to
make longer. In the context of the present invention this means to
increase the period of the circadian rhythm.

[0022]In another aspect, the invention provides use of a composition
comprising an inhibitor of adenylyl cyclase for the manufacture of a
medicament for a disorder of the circadian rhythm.

[0023]In another aspect, the invention provides use of a composition
comprising an inhibitor of adenylyl cyclase for the manufacture of a
medicament for jet lag.

[0024]In another aspect, the invention provides use of a composition
comprising an inhibitor of adenylyl cyclase for the manufacture of a
medicament for familial advanced sleep phase syndrome.

[0025]In another aspect, the invention provides rise of a composition
comprising an inhibitor of adenylyl cyclase for the manufacture of a
medicament for shift lag.

[0026]Preferably said inhibitor is a purine site ligand.

[0027]Preferably said inhibitor is specific for adenylyl cyclases.

[0028]Preferably said inhibitor does not act at the
Gs.sub.α-binding-site of adenylyl cyclase.

[0029]Preferably said inhibitor acts at the P-site of adenylyl cyclase.

[0030]Preferably said inhibitor is selected from the group consisting of
9-(tetrahydrofuryl)-adenine, 9-(cyclopentyl)-adenine and
2',5'-dideoxyadenosine.

[0031]Preferably the inhibitor is 9-(tetrahydrofuryl)-adenine (SQ 22,536
or THFA) or 2',5'-dideoxyadenosine.

[0032]Preferably said inhibitor is 9-(tetrahydrofuryl)-adenine.

[0033]The composition may consist of the inhibitor of adenylyl cyclase.

[0034]In another aspect, the invention provides an adenylyl cyclase
inhibitor for use in the treatment of a disorder of the circadian rhythm.

[0052]Adenylyl cyclase (sometimes referred to as adenylate cyclase) is a
family of proteins. There are numerous different isotypes. These isotypes
can have differential tissue expression patterns.

[0053]There are numerous adenylyl cyclase inhibitors known in the art.
Different classes of inhibitor can have different points of action. It is
disclosed that Gsα acting inhibitors tend to reduce cAMP to
zero, or to undetectable levels. The effect of this drastic reduction is
ablation of the circadian rhythm. This leads to arrhythmic behaviour and
loss of the clock function. Following removal of the inhibitor, clock
function can be restored.

[0054]By contrast, purine-site (P-site) inhibitors reduce cAMP levels, but
do not completely ablate them. P(purine)-site ligands inhibit via a
mechanism preferably having at least one of the following features:
non-competitive; dead-end; post-transition state mechanism; specific for
adenylyl cyclases; preferably said inhibitors act via a mechanism having
all of said features. According to the present invention, P site adenylyl
cyclase inhibitors can be used to extend the clock period, i.e. to slow
the clock mechanism. Advantageously, P site inhibitors do not completely
remove clock function, but rather slow it in a controlled manner.
Preferably the inhibitors is a P-site inhibitor. Preferably the inhibitor
is not a Gsα-binding site inhibitor; preferably the inhibitor is
not a GaS site inhibitor (e.g. MDL--MDL antagonises the action of
forskolin (which acts at the Gsalpha site)). Preferably the inhibitor is
not a Gi site inhibitor (e.g. pertussis toxin--Pertussis toxin (PTX)
irreversibly ADP-ribosylates G_i and G_o proteins, inactivating them and
preventing their inhibition of adenylyl cyclase (AC). PTX treatment
affects amplitude and not period.).

[0055]P-site inhibitors and their properties are reviewed in Dessauer et
al (1999 TIPS vol 20 p 205). `P-site ligand` refers to a moiety which
binds at the AC catalytic (purinergic) site. A `P-site inhibitor` has
this binding property and inhibits enzyme activity.

[0056]An important property of p-site inhibitors is that they are not
competitive with respect to ATP (the enzymatic substrate).
Non-competitive/uncompetitive inhibitors affect the reaction rate, by
altering the stability of the enzyme substrate or enzyme-product complex.
In the case of AC, its rate is determined by two factors: 1) the rate of
the cyclisation reaction and 2) the rate of release of PPi (one of the
products--inorganic phosphate). P-site inhibitors bind to the purinergic
hydrophobic pocket (following dissociation of cyclic AMP) and stabilise
the conformation of the post-transition state enzyme-product complex,
thus slowing the release of PPi (see Dessauer et al ibid-incorporated
herein by reference), and leading to an accumulation of the enzyme-PPi
complex. Thus, p-site inhibitors form a dead-end complex by binding to
the active site of adenylate cyclase in the presence of pyrophosphate.

[0057]Determination of p-site binding is straightforward enzyme kinetics
and has been carried for numerous p-site inhibitors in the art, for
example as in Dessauer and Gilman (1997-JBC vol 272 pp 27787-95).

[0058]The skilled operator can carry out this assay and decide whether a
moiety binds the p-site of AC. In case any further guidance is needed, a
dissociation constant (Kd) of <100 uM would imply specificity.
However, clearly with inhibitors it may be more appropriate to assess Ki,
or IC50 or EC50 depending on operator choice. In any case it should be
noted that due to the mechanism by which p-site inhibitors associate with
AC, it is expected to only observe binding in the presence of Pi
(inorganic phosphate).

[0059]Regarding AC activity assay, and thus determination of inhibition,
this is a standard biochemical assay and can be performed in a number of
ways well known in the art. In case any guidance is needed, preferably
the AC assay is carried out as described in Onda et al (2001 JBC vol 276
pp 47785-47793, in particular first para R-col page 47786 which is
incorporated herein by reference). Activity/inhibition may be determined
for any suitable moiety such as the whole adenylyl cyclase, or purified
catalytic domain(s). Determination of inhibition may thus be made by the
skilled worker. In case any further guidance is needed, for an in vitro
assay it is expected that even weak inhibitors will have IC50<5 mM.

[0060]Lithium can elicit an increase in circadian period. However, a link
to AC inhibition by lithium is unproven, and unlikely. Indeed, lithium is
known in the art to be preferentially active against glycogen synthase
kinase, inositol monophosphatase, inositol polyphosphate 1-phosphatase,
glycogen synthase kinase-3, fructose 1,6-bisphosphatase, bisphosphate
nucleotidase, and phosphoglucomutase (all EC50<2 mM). By contrast,
lithium's physiological EC50 against adenylyl cyclase is reported to be
about 20 mM (Goldberg et al, 1988, Am J Renal Physiol), which is a
concentration verging on toxicity in humans. Study of a physiologically
relevant role of lithium, both in vivo and in vitro, supports the
prevailing idea in the art that any action of lithium on AC/cAMP is
likely to be indirect. Moreover, lithium's possible pharmacological
action on AC (in vitro) is mostly thought to be mediated by competing
with Mg2+ ions at the catalytic site, which is a general feature of its
mechanism of inhibition (as opposed to non-competitively with ATP, in the
case of preferred inhibitor of the invention THFA). As a consequence,
lithium is not viewed as a p-site inhibitor in the field, or indeed as
being at all specific for AC; lithium's mode(s) of action is considered
to be through competition with Mg2+. In addition, whilst 10 mM
lithium-elicits an increase in SCN period in vitro of 2-3 hours, it does
not affect circadian period in 3T3 fibroblasts. By contrast, treatment
according to the present invention such as with THFA increases period in
all tissues tested. Furthermore, it is widely believed in the art that
the period effects of lithium are mediated through inhibition of GSK3beta
(EC50=0.8 mM). Thus, lithium is not a p-site inhibitor of adenylyl
cyclase. Preferably the adenylyl cyclase inhibitor of the invention is
not lithium.

[0061]As disclosed herein, certain types of adenylyl cyclase inhibitor
produce a `complete` knock-down of cAMP levels. This is not desirable for
most aspects of the invention since it may ablate clock function or lead
to a complete resetting of the clock. Preferably the invention reduces
adenylyl cyclase activity without completely removing it. In this way,
the invention advantageously relates to extension of the period (rather
than removal of clock function). An example of a `complete` knock-down
adenylyl cyclase inhibitor is MDL-12,330A. An example of a preferred
inhibitor providing a beneficial reduction in adenylyl cyclase (but not a
`complete` knock-down) is THFA. In other words, the invention preferably
produces a low steady state of adenylyl cyclase activity (as effected by
THFA) rather than a `complete` knock-down (as effected by MDL).

[0062]Thus, in a broad aspect, the invention relates to the use of an
adenylyl cyclase inhibitor in a manipulation of the circadian clock.
Preferably, the invention relates to use of an adenylyl cyclase inhibitor
in extension of the period of the circadian clock.

[0063]Preferably the adenylyl cyclase inhibitor is not a Gsα
site inhibitor.

[0064]Preferably the adenylyl cyclase inhibitor is a P site inhibitor.

[0065]Preferably the adenylyl cyclase inhibitor is 9-tetrahydrofuryl
adenine (also referred to as `THFA` or `SQ22,536`);
2',5'-dideoxyadenosine, or 9-(cyclopentyl)-adenine.

[0067]Preferably the inhibitor is membrane permeable. Preferred P-site
inhibitors which are membrane-permeable are 9-(tetrahydrofuryl)-adenine
(SQ 22,536 or THFA), 2',5'-dideoxyadenosine, and 9-(cyclopentyl)-adenine.
All three have the advantage of being water soluble up to at least 125
mM. Preferably the inhibitor is 9-(tetrahydrofuryl)-adenine (SQ 22,536 or
THFA) or 2',5'-dideoxyadenosine. Preferably the inhibitor is
9-(tetrahydrofuryl)-adenine (SQ 22,536 or THFA).

[0068]Of the P-site inhibitors, THFA is the most preferred. It has the
advantage of being most readily water soluble. The other inhibitors are
less soluble (for some embodiments involving 2',5'-dideoxyadenosine it is
preferred to prepare stocks in DMSO prior to dilution).

[0069]9-(cyclopentyl)-adenine advantageously has greater metabolic and
chemical stability than THFA and is also water soluble. However, it may
be less potent than THFA and thus the dosing may need to be
correspondingly adjusted as taught herein.

[0070]Regarding adenylyl cyclase, this is an enzyme with many family
members. Different adenylyl cyclase isoforms have different expression
patterns. Without wishing to be bound by theory, the proposed mechanism
is that AC turnover is reduced in all tissues leading to a global slowing
down of the clock i.e. throughout the organism being treated. Given the
presence of different isoforms with different tissue expression patterns,
it may be that more widely expressed isoforms of AC are better targets
relative to those with restricted tissue expression. It may even be the
case that particular adenylyl cyclase isoform-s have a more prominent
circadian role. Nevertheless, advantageously adenylyl cyclase inhibitors
are used which have activity across the adenylyl cyclase isoforms,
thereby alleviating the need to use cocktails of isoform-specific
inhibitors. Preferably the inhibitor is a P-site inhibitor, preferably
THFA.

[0072]Strikingly, the circadian actions of cAMP disclosed herein are
protein kinase A-independent, but are dependent on the Epac family of
guanine-nucleotide exchange factors, signalling through Jun N-terminal
kinases (JNK) to activate circadian gene expression. Combination
inhibition, such as simultaneous inhibition of AC and JNK activities
causes unprecedented lengthening of circadian period in both SCN slices
and fibroblasts.

[0073]Thus, the present invention relates to combinations of the
treatments disclosed herein with inhibition of JNK, such as by
administration of inhibitors of JNK.

[0082]Semapimod from Cytokine PharmaSciences; A suitable dose is 8 or 25
mg/m(2) via the gut.

[0083]AM-111 from Xigen. A suitable dose is 0.4 mg/ml or 2 mg/ml of AM-111
in a 250 ul gel formulation. This has been administered for other
applications by transtympanic injection into the most affected ear.

[0088]The invention finds application in familial advanced sleep phase
syndrome (FASPS). Subjects suffering from FAPSS typically show a 21-22
hour rhythm. According to the present invention, this condition is
treated by administration of an adenylyl cyclase inhibitor in order to
slow their biological clock to a 24 hour rhythm. The dosage given is
preferably titrated in order to provide the lowest dose in order to
achieve an approximately two hour delay to their usual circadian period.

[0089]The invention finds application in the treatment of jet lag.
Typically, a body can adjust approximately one hour per cycle. Therefore,
if a subject is jet lagged by a time difference of approximately five
hours, the expectation is that it would take approximately five days for
their circadian rhythm to become properly adjusted to the new time zone
in which they are placed. According to the present invention, a subject
suffering from jet lag may be treated by administration of an adenylyl
cyclase inhibitor. The dose of the inhibitor would be adjusted by the
operator according to the length of delay needed in the circadian rhythm.
For example, a subject suffering jet lag after flying from London to New
York would typically need approximately five hours adjustment to their
cycle. Thus, the dose should be adjusted in order to provide an
approximately 29 hour cycle. A preferred dose for this 29 hour cycle is a
final serum concentration of 0.5 to 1 mM THFA--(depending on the
retention of THFA this may require 100 mg/kg intake for an adult human).
Preferably a treatment is provided during the circadian cycle in which
the flight is made. For larger adjustments, a dose may be provided in two
consecutive cycles. For example, for a subject flying from London to Los
Angeles, the time difference is greater than 6.5 hours (the approximate
maximum adjustment provided by a single dose) and therefore such a
subject would be in need of doses in two consecutive cycles. Preferably
the first dose is given in the cycle in which the flight is taken.

[0090]Preferably the invention is applied to Westward flights. This is
because the invention is preferably used in the delay or extension or a
circadian rhythm, i.e. a lengthening of a day, which is the effect
observed with Westward jet lag.

[0091]The invention may be applied for Eastward jet lag. In this
embodiment, the dose should be adjusted taking into account the need to
advance the clock. According to the present invention, such advances
should be treated as multiple retardations (multiple lengthenings) of the
circadian cycle so that a net effect produce can be equated to an advance
in the cycle. For example, if a subject flies East and experiences a
twelve hour shift in the day/night cycle, then preferably two doses of
adenylyl cyclase inhibitor would be given in two consecutive cycles,
producing approximately a six hour shift in each of those two cycles,
amounting to a twelve hour shift in total thereby synchronising the
subject's circadian rhythm with the local time. As will be appreciated in
this example, rather than advancing the subject's circadian clock, it has
been retarded in order to achieve the same effect i.e. synchronisation of
their clock with the new local time. This principle should be applied
when operating the present invention to "advance" a subject's clock. In
particular, combination treatments such as adenylyl cyclase inhibitor
given with JNK inhibitor, are particularly suitable for Eastward jet tag
applications. This is because the synergistic extra lengthening of the
period using such combinations can produce A effect which can be equated
to the advance of the clock as explained above. For example, giving a
dual treatment with adenylyl cyclase inhibitor and JNK inhibitor can
extend the period to approximately 36 hours; this will have an effect
comparable to a 12-hour advance and therefore advantageously reduces the
number of single treatments (and cycles) which might otherwise be
required to produce the same adjustment (e.g. by multiple smaller
retardations).

[0092]The invention may be applied to the treatment of shift workers. For
example, when a worker embarks upon a series of night shifts, their clock
may be adjusted by administration of adenylyl cyclase inhibitor in
advance of the first night shift. This will extend their circadian cycle
so that they could delay their metabolism, sleep, and other cycles, until
near the end of their shift. If their shift exceeds approximately 6.5
hours (approximately the largest change available in a single cycle) then
the close can be divided over two or more consecutive cycles as
appropriate to achieve the necessary change in rhythm. Alternatively,
shift workers may take doses on an ad hoc basis in order to adjust their
clock ahead of a changing shift pattern.

Combinations

[0093]Lithium treatment has been shown to lengthen circadian period.
Lithium treatment typically lengthens the period to approximately 26
hours in organotypic slices, and to approximately 25 hours in whole
animals. Without wishing to be bound by theory, it is believed that
adjustment of the clock using lithium operates via a different mechanism
to adjustment of the clock using adenylyl cyclase inhibitors. Thus, in
one embodiment, lithium treatment may be advantageously combined with
treatment using adenylyl cyclase inhibitors to produce an additive
effect, which may advantageously reduce the number of administrations
required to achieve a certain magnitude of adjustment.

[0094]Lithium is preferably used at 10 mM, preferably 3 mM, preferably at
established doses for clinical use of lithium in humans such as 0.8-1.2
mM final serum concentration.

[0095]Melatonin has been used in, the prior art in the adjustment of
circadian rhythm. Melatonin typically produces an acute phase shift of
the circadian rhythm. Thus, in one embodiment, the invention relates to
the administration of melatonin together with an inhibitor of adenylyl
cyclase. In this way, the rhythm may be advantageously phase shifted and
delayed in combination in order to achieve the desired adjustment.

[0096]Melatonin is preferably used at maunfacturer's recommended doses.
Preferably melatonin is used at a dose of about 0.5-5 mg/day (i.e. per
cycle) for an average adult human.

[0097]Jun N-terminal kinase (JNK) inhibitors can be used to extend the
period of the circadian rhythm. Indeed; it is disclosed herein for the
first time that JNK inhibitors can act synergistically with adenylyl
cyclase inhibitors according to the present invention to produce a still
further enhanced extension to the period of the circadian rhythm. This is
particularly useful in applications such as eastward jet lag.

[0098]In another embodiment, the invention relates to a triple combination
of lithium, melatonin and an adenylyl cyclase inhibitor which
advantageously maximises the size of the adjustment which can be made in
any one circadian cycle.

Administration

[0099]In principle, multiple small adjustments to circadian cycle are
preferable to a single large adjustment. An advantage of making multiple
smaller adjustments is to ease the impact on the patient of making a
single large adjustment in a particular given cycle. Thus, if a six hour
adjustment was required then preferably two three hour treatments would
be provided in two consecutive cycles.

[0100]Preferably a single cycle is adjusted by 2-12 hours, preferably by
2-10 hours, preferably by 2-8 hours, preferably by 2-6 hours preferably
by 2-4 hours, preferably by 3 hours per cycle, preferably by 2 hours per
cycle.

[0101]In general, smaller doses are used for smaller changes in period,
and vice versa. Highest shifts are observed in NIH 3T3 fibroblasts. These
have a circadian period of about 21 hours. In 1.2 mM THFA they shift to
about 30 hours. Thus the probable maximum period increase in these
conditions is approximately 9 hours. The suprachiasmatic nucleus (as the
master clock) has a longer intrinsic period of 24 hours, and so shifts by
approximately 6 hours under similar conditions.

[0102]Preferably administration is oral administration.

Dose Levels

[0103]Typically, a physician will determine the actual dosage which will
be most suitable for an individual subject. The specific dose level and
frequency of dosage for any particular patient may be varied and with
depend upon a variety of factors including the activity of the specific
compound employed, the metabolic stability and length of action of that
compound the age, body weight, general health, sex, diet, mode and time
of administration, rate of excretion, drug combination, the severity of
the particular condition, and the individual undergoing therapy.

[0104]Preferably THFA is orally administered, preferably in water. For
mice, the concentration at administration is 4 mM in water.

[0105]Alternatively THFA may be administered in tablet form, preferably
tablet form is used for humans.

[0106]As with any dosing, regime, attention must be paid by the operator
to choice of an appropriate dose of THFA for chronic intake, balancing
the rate at which it is metabolised/excreted. Furthermore, attention must
be paid to what time of day the drug is administered (e.g. as would be
the case in acute treatment) in considering any effects on the response
to/efficacy of the drug (in circadian terms, attention should be paid to
the Phase Response Curve as necessary).

[0107]Depending upon the need, the agent may be administered at a dose of
from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more
preferably from 0.1 to 1 mg/kg body weight.

[0108]For SQ22,536 (THFA), a typical dose is 50 mg/kg for an adult human.

[0109]For 2',5'-dideoxyadenosine, a typical dose is 60 ng/kg for an adult
human.

[0110]For 9-(cyclopentyl) adenine, a typical dose is 75 mg/kg for an adult
human.

[0111]The dose may preferably be split into two or more applications for
administration in two or more separate cycles in order to advantageously
reduce the impact of large changes in rhythm produced over a single
cycle.

[0112]Within a given cycle, preferably the dose is given in a single
administration.

[0113]Preferably the dose is given orally or by injection, preferably
orally.

[0114]Depending upon the time delay desired for a given cycle, the dose of
adenylyl cyclase inhibitor is varied accordingly. For example, to achieve
maximum delay (of approximately 6.5 to 9 hours, preferably 6.5 hours for
a whole animal), a dose of approximately 100 mg/kg of THFA would be
administered to an average adult male human. To achieve a delay of
approximately 3 hours, the dose would be approximately 30 mg/kg.

[0115]Treatment with 0.3% lithium lengthens period by approximately 2%.

[0116]Treatment with 25% D2O increases period by approximately 5%.

Pharmaceutical Compositions

[0117]The present invention relates to compositions comprising an
inhibitor of adenylyl cyclase, and to uses of those compositions. In some
embodiments the invention relates to uses of the inhibitor of adenylyl
cyclase itself.

[0118]The present invention also provides a pharmaceutical composition
comprising a therapeutically effective amount of an adenylyl cyclase
inhibitor of the present invention and a pharmaceutically acceptable
carrier, diluent or excipient (including combinations thereof).

[0119]The pharmaceutical compositions may be for human or animal usage in
human and veterinary medicine and will typically comprise any one or more
of a pharmaceutically acceptable diluent, carrier, or, excipient.
Acceptable carriers or diluents for therapeutic use are well known in the
pharmaceutical art, and are described, for example, in Remington's
Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
The choice of pharmaceutical carrier, excipient or diluent can be
selected with regard to the intended route of administration and standard
pharmaceutical practice. The pharmaceutical compositions may comprise
as--or in addition to--the carrier, excipient or diluent any suitable
binder(s), lubricant(s), suspending agent(s), coating agent(s),
solubilising agent(s).

[0120]Preservatives, stabilizers, dyes and even flavoring agents may be
provided in the pharmaceutical composition. Examples of preservatives
include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
Antioxidants and suspending agents may be also used.

[0121]There may be different composition/formulation requirements
dependent on the different delivery systems. By way of example, the
pharmaceutical composition of the present invention may be formulated to
be administered using a mini-pump or by a mucosal route, for example, as
a nasal spray or aerosol for inhalation or ingestable solution, or
parenterally in which the composition is formulated by an injectable
form, for delivery, by, for example, an intravenous, intramuscular or
subcutaneous route. Alternatively, the formulation may be designed to be
administered by a number of routes.

[0122]Where the agent is to be administered mucosally through the
gastrointestinal mucosa, it should be able to remain stable during
transit though the gastrointestinal tract; for example, it should be
resistant to proteolytic degradation, stable at acid pH and resistant to
the detergent effects of bile.

[0123]Where appropriate, the pharmaceutical compositions can be
administered by inhalation, in the form of a suppository or pessary,
topically in the fold of a lotion, solution, cream, ointment or dusting
powder, by use of a skin patch, orally in the form of tablets containing
excipients such as starch or lactose, or in capsules or ovules either
alone or in admixture with excipients, or in the form of elixirs,
solutions or suspensions containing flavouring or colouring agents, or
they can be injected parenterally, for example intravenously,
intramuscularly or subcutaneously. For parenteral administration, the
compositions may be best used in the form of a sterile aqueous solution
which may contain other substances, for example enough salts or
monosaccharides to make the solution isotonic with blood. For buccal or
sublingual administration the compositions may be administered in the
form of tablets or lozenges which can be formulated in a conventional
manner.

[0124]For some embodiments, the agents and/or growth factors of the
present invention may also be used in combination with a cyclodextrin.
Cyclodextrins are known to form inclusion and non-inclusion complexes
with drug molecules. Formation of a drug-cyclodextrin complex may modify
the solubility, dissolution rate, bioavailability and/or stability
property of a drug molecule. Drug-cyclodextrin complexes are generally
useful for most dosage forms and administration routes. As an alternative
to direct complexation with the drug the cyclodextrin may be used as an
auxiliary additive, e.g. as a carrier, diluent or solubiliser. Alpha-,
beta- and gamma-cyclodextrins are most commonly used and suitable
examples are described in WO-A-91/11172, WO-A-94/02518 and WO-A-98/55148.

[0125]If the adenylyl cyclase inhibitor is a protein, then said protein
may be prepared in situ in the subject being treated. In this respect,
nucleotide sequences encoding said protein may be delivered by use of
non-viral techniques (e.g. by use of liposomes) and/or viral techniques
(e.g. by use of retroviral vectors) such that the said protein is
expressed from said nucleotide sequence.

[0126]In a preferred embodiment, the pharmaceutical of the present
invention is administered orally. Hence, preferably the pharmaceutical is
in a form that is suitable for oral delivery.

[0128]The components of die present invention may be administered alone
but will generally be administered as a pharmaceutical composition--e.g.
when the components are is in admixture with a suitable pharmaceutical
excipient, diluent or carrier selected with regard to the intended route
of administration and standard pharmaceutical practice. For example, the
components can be administered (e.g. orally or topically) in the form of
tablets, capsules, ovules, elixirs, solutions or suspensions, which may
contain flavouring or colouring agents, for immediate-, delayed-,
modified-, sustained-, pulsed- or controlled-release applications.

[0130]Solid compositions of a similar type may also be employed as fillers
in gelatin capsules. Preferred excipients in this regard include lactose,
starch, a cellulose, milk sugar or high molecular weight polyethylene
glycols. For aqueous suspensions and/or elixirs, the agent may be
combined with various sweetening or flavouring agents, colouring matter
or dyes, with emulsifying and/or suspending agents and with diluents such
as water, ethanol, propylene glycol and glycerin, and combinations
thereof.

[0132]It is to be understood that not all of the components of the
pharmaceutical need be administered by the same route. Likewise, if the
composition comprises more than one active component, then those
components may be administered by different routes.

[0133]If a component of the present invention is administered
parenterally, then examples of such administration include one or more
of: intravenously, intra-arterially, intraperitoneally, intrathecally,
intraventricularly, intraurethrally, intrasternally, intracranially,
intramuscularly or subcutaneously administering the component; and/or by
using infusion techniques.

[0134]For parenteral administration, the component is best used in the
form of a sterile aqueous solution which may contain other substances,
for example, enough salts or glucose to make the solution isotonic with
blood. The aqueous solutions should be suitably buffered (preferably to a
pH of from 3 to 9), if necessary. The preparation of suitable parental
formulations under sterile conditions is readily accomplished by standard
pharmaceutical techniques well-known to those skilled in the art. As
indicated, the component(s) of the present invention can be administered
intranasally or by inhalation and is conveniently delivered in the form
of a dry powder inhaler or an aerosol spray presentation from a
pressurised container, pump, spray or nebuliser with the use of a
suitable propellant, e.g. dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluroalkane
such as 1,1,1,2-tetrafluoroethane (HFA 134A®) or
1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA®), carbon dioxide or other
suitable gas. In the case of a pressurised aerosol, the dosage unit may
be determined by providing a valve to deliver a metered amount. The
pressurised container, pump, spray or nebuliser may contain a solution or
suspension of the active compound, e.g. using a mixture of ethanol and
the propellant as the solvent, which may additionally contain a
lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for
example, from gelatin) for use in an inhaler or insufflator may be
formulated to contain a powder mix of the agent and a suitable powder
base such as lactose or starch.

[0135]Alternatively, the component(s) of the present invention can be
administered in the form of a suppository or pessary, or it may be
applied topically in the form of a gel, hydrogel, lotion, solution,
cream, ointment or dusting powder. The component(s) of the present
invention may also be dermally or transdermally administered, for
example, by the use of a skin patch. They may also be administered by the
pulmonary or rectal routes. They may also be administered by the ocular
route. For ophthalmic use, the compounds can be formulated as micronised
suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as
solutions in isotonic, pH adjusted, sterile saline, optionally in
combination with a preservative such as a benzylalkonium chloride.
Alternatively, they may be formulated in an ointment such as petrolatum.

[0136]For application topically to the skin, the component(s) of the
present invention can be formulated as a suitable ointment containing the
active compound suspended or dissolved in, for example, a mixture with
one or more of the following: mineral oil, liquid petrolatum, white
petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound,
emulsifying wax and water. Alternatively, it can be formulated as a
suitable lotion or cream, suspended or dissolved in, for example, a
mixture of one or more of the following; mineral oil, sorbitan
monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60
cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.

Pharmaceutical Combinations

[0137]The agent of the present invention may be administered with one or
more other pharmaceutically active substances. By way of example, the
present invention covers the simultaneous, or sequential treatments with
an adenylyl cyclase inhibitor according to the present invention and one
or more steroids, analgesics, antivirals or other pharmaceutically active
substance(s) such as lithium and/or melatonin.

[0138]In particular, the present invention relates to the simultaneous, or
sequential treatments with an adenylyl cyclase inhibitor according to the
present invention and one or more JNK inhibitors.

[0139]It will be understood that these regimes include the administration
of the substances sequentially, simultaneously or together.

[0140]The therapy can include the treatment of one or more of those
disorders mentioned herein, or related complaint.

The Circadian Clock

[0141]Circadian rhythms are tractable and robust. They are known to
regulate behaviour, physiology and metabolism such as serum cortisol
levels, body temperature, sleep patterns and other biologically
significant characteristics. Circadian rhythms are typically observed to
be within a range 23.5-24.5 hours. The rhythms are manifest at the whole
organism level, the tissue level and even at the cellular level.

[0142]Oscillation of the circadian clock (occasionally referred to as the
`biological clock`) has two key properties. The first is the period. This
is the peak to peak (or trough to trough) time taken per cycle. In other
words, this is the time taken for one complete "revolution" of the clock.
In normal subjects, this is typically 24 hours. The second feature of the
oscillation is its amplitude. This refers to the maximum reach or maximum
values of the peaks and troughs of the cycling of the clock. The
amplitude and the period are separate features of the oscillations of the
clock. The present invention is primarily concerned with manipulation of
the circadian rhythm/clock, that it to say with manipulation of the
period of the rhythm/clock. Extension or elongation of the period of the
rhythm/clock may for simplicity be simply referred to as extension or
elongation of the rhythm/clock.

[0143]The circadian clock was originally thought to reside entirely in the
suprachiasmatic nucleus (SCN). Removal of the SCN resulted in arrhythmic
animals. However, more recently it has been observed that peripheral
tissues display their own intrinsic oscillators, although these are
typically less robust and of a lower amplitude, and under overall control
of the SCN. Furthermore, circadian rhythms of gene expression have been
detected in most cell types with up to 10% of the genome beg affected.
These phenomena are also thought to be under the direct or indirect
control of the SCN in order to achieve synchrony.

[0144]The accepted genetic model of the operation of the circadian rhythm
is outlined below. Briefly, an activating transcriptional complex
(including factors such as clock and bmal1) drives the transcription of
inhibitory proteins such as Per1/2/3, Cry1/2 during early circadian day.
These proteins then require many hours before they can stably be imported
into the nucleus. This is partially explained by the observation that
cytosolic phosphorylation of these proteins (by kinases such as GSK-3
etc) drives ubiquitin-mediated degradation of the negative limb clock
factors. However, they also appear to be stabilised by complex formation
with/phosphorylation by other kinases which eventually licenses the
nuclear entry of the inhibitory complexes, where they are able to repress
their own transcription. By the time these complexes have been
disassembled and the cycle can begin again almost exactly 24 hours have
passed. This transcriptional/translational feedback loop has additional
targets, so-called "output clock genes", and indeed a large number of
genes have a circadian pattern to their transcriptional or translational
profile. Every organism needs to reset its internal clock from external
cues each day. One of the signalling mechanisms for communicating phase
changes may be through 2nd messengers such as cAMP and/or calcium. One
potential effector is the presence of activatable CREs in several
circadian gene promoters. It is interesting to note that, of the clock
genes, the only mutants which completely lose rhythmicity are those for
PER2 and BMAL1. These are therefore currently regarded as essential
components of the clock in this model.

[0145]Moreover, studies in cyanobacteria show that recombinant circadian
proteins can sustain stable circadian cycles of auto-phosphorylation in
vitro, in the absence of transcription, adding further complexity and
confusion to the prior art view.

[0147]It is disclosed herein for the first time that cAMP signalling
constitutes a new level of circadian regulation.

[0148]Cyclic nucleotides have been extensively studied as second
messengers of intracellular events initiated by activation of many types
of hormone and neurotransmitter receptors. Receptors that stimulate the
conversion of ATP to cyclic 3',5'-adenosine monophosphate (cAMP) are
associated with G proteins. Binding of the hormone or neurotransmitter to
its membrane-bound receptor induces a conformational change in the
receptor that leads to activation of the α-subunit of the G
protein. The activated Gs subunit stimulates, while the Gi
subunit inhibits-adenylyl cyclase (AC), furthermore some AC isotypes are
known to be additionally regulated by intracellular calcium and/or
protein kinase C (PKC). Stimulation of AC catalyzes the conversion of
cytoplasmic ATP to cAMP. cAMP activates cAMP-dependent protein kinases,
and other effectors, including protein kinase A, (PKA). By catalyzing the
phosphorylation (activation or deactivation) of intracellular enzymes,
cAMP-dependent kinases elicit a wide array of metabolic and functional
processes. Negative regulation can occur in the pathway when
phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP to
adenosine-5'-monophosphate (5'-AMP). Several families of
phosphodiesterases (PDE-I-VI) act as regulatory switches by catalyzing
the degradation of cAMP to adenosine-5-monophosphate (5'-AMP). PDE II is
a low affinity PDE that can cleave both cAMP and cGMP. The activity of
PDE II is stimulated by cGMP. PDE III is a low affinity PDE that is
inhibited by cGMP and is involved in the regulation of smooth muscle and
cardiac contraction. PDE IV is highly selective for cAMP and is the high
affinity PDE present in most cell types.

[0149]We disclose for the first time that cAMP signalling pathways
constitute a core component of the mammalian circadian clockwork. First,
they sustain tissue and cellular rhythimcity in the circadian pacemaker
of the suprachiasmatic nucleus (SCN). Second, they mediate inter-cellular
synchronisation between SCN neurons. Third, they determine the intrinsic
period of circadian pacemaking both in vitro and in vivo. We demonstrate
that pharmacological inhibition of adenylyl cyclase (AC) leads to
dramatic lengthening of period in the SCN. These roles of cAMP signalling
in circadian timekeeping are general, also being evident in peripheral
mammalian tissues and cell lines. According to the invention, it is
believed that daily activation of cAMP signalling sustains progression of
the transcriptional cycle. These findings reveal a novel and
unanticipated point of circadian regulation in mammals, qualitatively
different from the prior published transcriptional feedback model. We
disclose novel therapeutic targets for circadian dysfunction.

[0150]It is disclosed herein for the first time that circadian adenylyl
cyclase activation is essential for setting a 24 hour period. Inhibition
of adenylyl cyclase leads to unprecedented period lengthening to as much
as 30 or 31 hours, or even more.

[0151]The view in the art regarding operation of circadian rhythms
involves a gene expression/protein feedback system, with second
messengers such as cAMP being mere effectors. However, it is surprisingly
disclosed herein that in fact cAMP is an essential element of the
circadian clock, governing the period. This surprising discovery has
allowed the present invention to be based on the manipulation of cAMP
levels in order to manipulate the period of the clock.

[0152]The definitive test whether a candidate entity is a structural part
of the clock mechanism is to assess period (i.e. length of cycle of the
clock) under different conditions for said entity. Merely assessing an
output or effector function is less rigorous and is therefore less
preferred. Preferably the effect of a particular intervention on period
is determined as indicative of an effect on the actual circadian clock
mechanism.

[0153]The fact that second messenger such as calcium levels rise before
firing suggests regulated process, more than just output, and led to the
inference that it is an essential feature, which is supported by
EDTA/BAPTA analysis. Thus, it is disclosed that cAMP is involved in
circadian rhythm regulation. This is in contrast to the prior art view of
it as a (non-essential) resetting mechanism--it had not been considered
before the present invention that cAMP is playing a central role in
generating circadian rhythms.

Further Applications

[0154]The invention may be applied in the modulation of effects of other
drugs. For example, the liver follows a physiological cycle controlled by
the circadian rhythm. The activities of the liver in the day are
different to those in the night. As a consequence, particular therapeutic
agents may be more rapidly or more slowly degraded by the liver following
administration at different times during a circadian cycle. Thus, the
invention finds application in the manipulation of the circadian period
followed by administration of a drug. This advantageously provides the
best efficacy for a given drug according to the point in a circadian
cycle at which it is administered. Thus, the invention may be used to
modulate the response to particular drugs by modulation of the circadian
rhythm.

[0155]It is well-known in the art that there is a daily peak in blood
pressure at the start of the day. This peak in blood pressure has been
correlated with higher incidences of stroke, cerebral infarction or
coronary attacks. Incidence of these conditions can be up to 30% higher
around the time of the daily peak in blood pressure. Thus, it is
desirable to target blood pressure controls to the early morning blood
pressure peak in order to combat this effect. Thus, the invention finds
application in manipulation of the circadian rhythm in order to modulate
early morning peaks in blood pressure and thereby, reduce the risk of
blood pressure induced disorders such as those above.

[0156]The invention finds application in space travel. The day length on
Earth is 23 hours and 56 minutes. Day length on Mars is 24 hours and 37
minutes. Thus, astronauts travelling to Mars need to adjust their
circadian rhythms in order to suit the increased Martian day length
relative to the day length of Earth. Thus, in one aspect the invention
relates to extension of day length to approximately 24 hours 37 minutes
by administration of an appropriate dose of adenylyl cyclase inhibitor to
a subject in need of same. Thurs, according to the present invention
travellers to Mars can increase the length of their circadian period to
match that of the local environs, thus avoiding adverse-effect(s) of
trying to live an Earth length day, on an alien planet.

[0157]The invention also relates to increasing circadian period length.
Dose dependant period length increase, preferably in all tissues, enables
Sufferers of Familial Advanced Sleep Phase Syndrome can be treated,
bringing their period to 24 hours (chronic treatment). Shift workers can
reduce the unpleasant side-effects and long term adverse health
consequences of keeping unusual hours by adjusting their circadian phase
to match their work day requirements through acutely manipulating their
circadian period length. Sufferers from "jet lag" can reduce the
unpleasant side-effects and long term adverse health consequences of
changing time zone by adjusting their circadian phase to match the time
zone of their destination through acutely manipulating their circadian
period length.

[0158]The invention also relates to treatment of Seasonal Affective
Disorder (SAD). Dose dependant period length increase, preferably in all
tissues, enables sufferers from SAD (or winter depression) to increase
their physiologically perceived duration of day length, and thus
alleviate the symptoms of SAD which accompany day length shortening at
higher latitudes.

[0159]The invention also relates to treatment of sleeping disorders. Dose
dependant period length increase, preferably in all tissues, enables
sufferers from insomnia and/or narcolepsy to have a means of coping with
their condition through manipulation of physiologically perceived daytime
and/or night time onset.

[0160]The invention also relates to treatment of depression. Lithium has a
characterised role in treating depression. Its major pharmacological
effect is mediated by inhibition of Glycogen Synthase Kinase 3β,
this is leads to an modest increase in circadian period (˜30
minutes in mouse behavioural rhythms, ˜2 hours in organotypic
tissue extracts). Because the SCN (suprachiasmatic nucleus--the master
clock in mammals) has extensive reciprocal connections with the 5-HT
centre the Median Raphe, and 5-HT treatment of SCN slices (in vitro)
leads to p shifts, treatment which also increases circadian period in the
SCN may advantageously have an anti-depressant action.

[0161]The invention also relates to treatment of appetite control. cAMP is
an important second messenger in signalling the fasting state
intracellularly, in pharmacological interference with this signalling
pathway, and advantageously may have an action in suppressing appetite,
particularly as the SCN has extensive communication with the orexin
neurons of the hypothalamus.

[0162]The invention also relates to modulation of action of other
drugs/treatments. Many drugs and healthcare treatments have been shown to
have variable efficacy depending on the time of circadian day/night at
which they are administered. Manipulation of a patient's physiological
day/night length may advantageously extend the therapeutic window for
maximal efficacy and/or reduce toxic side effects of other drugs.

[0163]The invention also relates to treatment of disordered sleep patterns
in neurodegenerative diseases. Patients with advanced Parkinson's,
Alzheimer's etc have disordered sleep patterns which makes them difficult
to care for without 24-hour supervision. Manipulation of physiologically
perceived day/night length may reduce these symptoms.

[0164]The invention may also relate to lifestyle/performance enhancement
applications. For example, an increase in physiological night duration
through acute treatment could help alleviate conditions such as sleep
deprivation. An increase in physiological day duration through acute
treatment could increase period of alertness, athletic performance, or
productivity.

[0165]In a preferred embodiment when the adenylyl cyclase inhibitor is
THFA, the invention also embraces treatment of blood pressure problems
such as high blood pressure. cAMP is a key regulator of
excitation-contraction coupling in the heart; this role is mainly
mediated by PKA, thus the use of a non-competitive P-site inhibitor may
advantageously reduce high heart rates.

[0166]In a preferred embodiment when the adenylyl cyclase inhibitor is
THFA, the invention also embraces treatment of specific tissues. THFA is
a membrane-permeable inhibitor; as such it may diffuse into multiple
tissues, preferably equally well regardless of tissue type. By the
addition of certain moieties to the THFA molecule, it may be targeted to
specific tissues. e.g. non-blood brain permeable moieties would affect
peripheral tissues only.

[0167]In a preferred embodiment when the adenylyl cyclase inhibitor is
THFA, the invention also embraces cancer treatment, where THFA may have a
tumour suppressor action.

[0168]In a preferred embodiment when the adenylyl cyclase inhibitor is
THFA, the invention also embraces stroke treatment. THFA may have an
anti-apoptotic role, thus administration following the occurrence of a
stroke may reduce brain damage.

[0169]Although Gsα site adenylyl cyclase inhibitors are not useful
in extension of the period of circadian rhythm according to the present
invention, they may advantageously be applied in other aspects as noted
herein.

[0170]The invention will now be described by way of examples which are
intended to be illustrative rather than limiting in nature. In the
examples, reference is made to the following figures:

[0202](a) Representative double-plotted, actograms of (left) vehicle- and
(right) THFA-treated mice entrained to a photoschedule of 12 hours light
(shaded) and 12 hours dark and fitted with osmotic mini-pump and central
cannula directed at SCN (asterisk) Mice were then released into
continuous dim-red light and free-running period determined Note longer
period in THFA-treated mice.

[0203](b) Group data (mean+SEM) reveal significant (p<0.01, t-test)
lengthening of free-running period in vivo by THFA compared to vehicle.

[0207](c) Representative traces show that over-expression of Epac
inhibitor Rap1S17N (red-curve) dampens circadian gene expression rhythms
in NIH 3T3 cells transfected with mBmal1::luc, both before and after a
medium change (arrow). Over-expression of wild-type Rap (blue) or empty
vector (black) has no effect.

[0208](d) Representative traces show suppression of circadian gene
expression in 3T3 cells-transfected with mBmal1::luc reporter and treated
with interfering RNA to either Epac1 (blue) or Epac 2 (red) compared to
empty vector (black).

[0211]FIG. 19 shows a schematic model of how the circadian clockwork is a
product of interlinked AC-dependent signalling pathways and
transcriptional feedback loops.

[0212]The canonical feedback oscillator of the clock involves
auto-regulatory feedback loops (green) driven by periodic, alternating
activation and inhibition at DNA regulatory sequences such as E-boxes and
ROREs. Transcriptional output from the loops is translated into various
extra-cellular signals that sustain circadian biology. The circadian
transcriptional loops are sustained by adenylyl cyclase (AC) signalling,
via Epac and JNK (red)-likely acting through AP-1 DNA regulatory
sequences in clock genes. Circadian cycles of AC activity are a product
of output from the intra-cellular transcriptional loops (e.g. cyclical
expression of AC) and afferent stimuli acting upon Gsα and Gsi
(blue). Interference with either the transcriptional feedback loop or AC
signalling can either suspend the clockwork or alter its period. Within
the circuitry of the SCN, two further special conditions apply. First,
the extra-cellular output of one cell in the form of VIP neurosecretion
will provide synchronising and sustaining cues to post-synaptic cells.
Second, retinal innervation mediated by glutamatergic cues can
synchronise clock cells by resetting the transcriptional feedback loop
via [Ca++]i-dependent CRE DNA regulatory sequences. In tissues other than
SCN, AC and [Ca++]i-dependent pathways will be addressed by alternative,
tissue-specific synchronising actors, and likely involve extensive
interaction between signalling cascades.

[0218](e) Addition of THFA to NIH 3T3 cells, suppresses peak levels but
maintains basal levels of cAMP (mean+SEM, n=3 for all, control data as in
(a)).

[0219](f) Circadian gene expression patterns in fibroblast cultures
parallel to those in (e) but transfected with mBmal1::luc show
prolongation of circadian period is associated within loss of peak cAMP
titres.

[0232]Studies were licensed under the UK Animals (Scientific Procedures)
Act 1986, with Local Ethical Review by the Medical Research Council and
the University of Cambridge. Per1::luciferase and; Per2-luc transgenic
mice were used. For organotypic slice culture, brains were removed from
pups 5-10 days old and sectioned at 300 um with a McIlwain "Tissue
Chopper." Slices were sorted and trimmed to contain principally SCN
tissue and placed onto a Millipore membrane insert (PICMORG) for culture
at 37° C. in 5% CO2 as described previously (Gainer et al, 1998).
For long-term recordings, slices were transferred to 1.1 ml HEPES
buffered medium with 100 uM beetle luciferin (Promega) in a
glass-bottomed Petri dish sealed with a coverslip and vacuum grease.
Total bioluminescence was recorded with Hamamatsu photomultiplier tube
assemblies housed in a light-tight 37° C. incubator.
Photomultiplier recordings were expressed as counts per second integrated
over 6 min sample bins. Periods are peak-to-peak averages for not less
than 3 cycles, with at least 3 replicates for each data point. cAMP
assays were determined by R&D systems 3rd generation cAMP ELISA kit. All
drugs were purchased from Sigma Aldrich, made up as a stock solution in
medium or DMSO; then added to tissue medium.

[0233]Circadian timing in mammalian cells is based upon an auto-regulatory
transcriptional/post-translational feedback loop, pivoted around the
rhythmic expression of Period and Cryptochrome genes. Although circadian
activation of various second-messenger signalling cascades (including
cyclic nucleotides, MAPK and calcium) has been widely observed, their
role within the clockwork has been viewed primarily in respect of
entrainment, most obviously induction of Per expression. In VIP2 receptor
knockout mice (Vip2r-/-), however, the molecular clockwork is suspended
in most suprachiasmatic nucleus (SCN) neurons. This receptor signals via
adenylyl cyclase (AC). We therefore sought to test the role of AC
signalling in maintaining circadian time-keeping in mammals, using
real-time bioluminescent recording of circadian gene expression.

Experimental

[0234]Inhibition of the Gsα-binding site of AC caused a
dose-dependent, reversible, dampening of circadian gene expression from
Per1::luciferase organotypic SCN slice cultures, monitored by
photomultiplier tubes. To determine the generality of this effect, we
examined 3-T3 fibroblast cells. Abrogation of the endogenous oscillation
of cAMP concentration stopped circadian gene expression as reported by
Bmal1::luciferase reporter constructs P-site AC inhibition prolonged
fibroblast period from ca. 21 hours to ca. 30 hours. Comparable treatment
of Per1::luciferase SCN slices lengthened circadian period up to 30
hours.

[0235]Loss of cAMP signalling may account for the loss of molecular
timekeeping in the SCN of the Vip2r-/- mutant mouse. More generally, cAMP
signalling pathways are essential to sustain, mid regulate period of, the
mammalian circadian clockwork, both in SCN and in peripheral cells. Thus
it is demonstrated according to the present invention that P-site
adenylyl cyclase inhibition prolongs the circadian period.

Example 2

Demonstration of Prolongation of Circadian Rhythm Using Adenylyl Cyclase
Inhibitors

[0236]We disclose the cyclic AMP (cAMP) pathway as a new mode of
manipulating circadian rhythms in mammals. We show that observed
twice-daily, intracellular changes in cAMP levels are an essential
feature of the clock, rather than an output. Our manipulation of cAMP
synthesis confirms this role. Furthermore, the invention identifies
previously characterised intracellular targets for this application.

[0237]Firstly, we, have found that treatment of a range of mouse tissues
(e.g. organotypic brain and kidney slices, fibroblasts etc) with common,
commercially available-inhibitors of the enzyme adenylyl cyclase-prolongs
circadian period (as detected by a range of bioluminescent reporters)
from ˜24 to >30 hours, without dampening them. This effect is
unprecedented in circadian physiology/pharmacology.

[0238]The preferred inhibitors according to the present invention belong
to a class historically called P(purine)-site ligands, which inhibit via
a non-competitive, dead-end, post-transition state mechanism which makes
them specific for adenylyl cyclases. The most effcacious of these
inhibitors is THFA (9-(Tetrahydrofuryl)-adenine), or SQ 22,536].

[0239]THFA is membrane-permeable, water-soluble and has previously safely
been used on live rodents via intracranial injection (e.g. Marks et al,
Neuroscience, 2000 ibid.). Furthermore, we have not observed any toxic
effects in vitro during chronic incubations with THFA for periods of up
to 7 days, at concentrations as high as 2 mM. Period reverts to its
normal value of approx. 24 hours soon after removal of the drug.

[0240]We have found that application of THFA to mouse fibroblasts reduces
the detectable, endogenous, biphasic rise in cAMP, without affecting its
nadir. Oral delivery of this drug to mice thus advantageously alters
their circadian period without compromising basal cAMP functions,
demonstrating the utility as treatment for disrupted, human circadian
rhythms.

[0245]VPAC mice are poorly rhythmic in their behaviour. This is reflected
in the poor rhythmicity and low amplitude SCN rhythms.
Electrophysiological measurements have shown that an additional phenotype
of VPAC SCN is that they are hyperpolarised. Thus, they can be
"kick-started" by K+, or AP-4 (sodium channel blocker). This temporally
restores synchrony and some amplitude to the slice.

[0246]This effect is likely mediated, at least partly by an extra-cellular
calcium flux, as it can be abrogated by pretreatment of the slice with
1.6 uM EGTA.

[0247]However, VPAC receptor is not an ion channel, but is actually a GPCR
which is understood to signal through Gs. We thus investigated matters
further by studying cAMP.

Cyclic Nucleotide Metabolism--cAMP

[0248]Cyclic nucleotides have been extensively studied as second
messengers of intracellular events initiated by activation of many types
of hormone and neurotransmitter receptors. Receptors that stimulate the
conversion of ATP to cyclic 3',5'-adenosine monophosphate (cAMP) are
associated with G proteins. Binding of the hormone or neurotransmitter to
its membrane-bound receptor induces a conformational change in the
receptor that leads to activation of the a-subunit of the G protein. The
activated Gs subunit stimulates, while the Gi subunit inhibits adenylyl
cyclase (AC). Stimulation of AC catalyzes the conversion of cytoplasmic
ATP to CAMP.

[0249]Cummings et al had suggested an essential role for cAMP (in a
biochemical model for circadian clocks) in 1975, but this idea was
subsequently disproved in Neurospora.

[0250]The current view in the alt is that cAMP involvement is limited to a
role in phase resetting.

Example 4

Role of Adenylyl Cyclase in Circadian Rhythm

Adenylyl Cyclase Agonist

[0251]Forskolin is an adenylyl cyclase agonist with a well-characterised
action.

[0252]FIG. 4 shows a graph and a bar chart of the effect of AC agonist.

[0253]Application of Forskolin restored some activity to VPAC slices.

[0254]The appearance is of mimicking the effects of high potassium, and
implying greater synchrony between neurons. However, as with potassium
treatment, the stronger rhythms do gradually deteriorate. This presumably
reflects the upregulation of intracellular PDEs, as rather than restore
the proposed circadian increase in cAMP, the treatment has made it
constuitively higher.

[0255]However, the initial first few cycles following application are
unambiguously closer to wild type rhythms, supporting the model disclosed
herein.

Adenylyl Cyclase Inhibitor

[0256]In mammals, there are at least ten distinct adenylyl cyclase
isozymes, all but one of which are membrane-bound and are central to one
of the most important transmembrane signal transduction pathways. The
soluble form is regulated by bicarbonate, whereas membrane-bound forms
are regulated by numerous neurotransmitters and hormones through cell
surface receptors linked via heterotrimeric (αβγ)
stimiulatory (Gs) and inhibitory (Gi) guanine nucleotide-dependent
regulatory proteins (G-proteins). Most isozymes are activated by
Gαs, but differ more significantly in their regulation by Gαi
and in the effects of Gβγ. These adenylyl cyclase isozymes
exhibit a putative topology with 12 membrane-spanning regions and two
˜40 kDa cytosolic domains (C1 and C2), one after each six
membrane-spanning region. C1 and C2 share large conserved regions that
interact to form a cleft forming the catalytic active site. N-terminus
domains are highly variable and serve regulatory roles. Activation by
Gαs occurs through its interaction with the C2 domain of adenylyl
cyclase yielding the active enzyme: GTPαsC. Inhibition by G
proteins may occur by a direct effect of Gαi with the C1 domain of
adenylyl cyclase or by the recombination of βγwith Gαs.

[0257]FIG. 5 shows a graph of cAMP timecourse. This experiment is in 3T3
cells. The effect can be seen in the figure.

[0258]MDL 12,330A is a potent, specific adenylyl cyclase inhibitor which
is membrane permeable and irreversibly binds the Gsα site with an
IC50 of 250 um.

[0264]In light of the fact that MDL dampened 3T3s using a different
promoter, we investigated whether cAMP signalling is a general
requirement for circadian rhythms in mammalian tissues.

[0265]Without wishing to be bound by theory, in peripheral tissue
endogenous generation of rhythmic second messenger signalling may be
essential to sustain rhythmicity, and in the SCN endogenous generation of
rhythmic second messenger signalling must be reinforced by rhythmic
extracellular stimuli to sustain high amplitude rhythms.

[0266]Thus we arrived at a modified view which can be summarised as
follows: [0267]VPAC2-/- mice have impaired circadian rhythms because
there is no Gs-stimulated rise in cAMP. [0268]cAMP rhythm is essential
for rhymnicity in the SCN. [0269]cAMP rhythm is essential for rhythmicity
in peripheral tissues. [0270]AC agonist has no period effect

[0273]Without wishing to be bound by theory, we propose that a cytosolic
clock may be involved with transcriptional rhythms as an output (eg.
cyanobacteria).

[0274]The prior alt model accounting for circadian rhythms involves a gene
expression/protein feedback system with cAMP signalling relegated to a
minor role in phase resetting. We present a fundamentally different new
view of circadian rhythms in which cAMP plays a far more central role.

Example 5

Manipulation of Circadian Rhythm

[0275]We show that circadian adenylyl cyclase activation is essential for
setting 24 hour period. We show that inhibition has no effect on the
amplitude, only on the period of oscillation. Me demonstrate a 30%
increase in period to 31 hours. This is the longest ever observed to date
in mammalian circadian biology

[0278]Even at increased doses there is still no significant amplitude
effect.

[0279]This is the longest observed mammalian circadian cycle. For
comparison, we have studied slices in 10 mM Lithium, which give a 26 hour
period.

[0280]It should be further noted that washout of the inhibitor restores
previous period. This demonstrates the utility of the invention in
adjusting circadian rhythm in the short term whilst advantageously
avoiding deleterious permanent or long-term extension of the period
following a single treatment. In other words, once the subject ceases to
take in the active compound in accordance with the invention, their
rhythm returns to normal period. Thus, for jet lag, shift lag or related
applications then a short-term course of treatment is appropriate to
adjust the rhythm which then reverts to approx. 24 hour period. Equally,
for FASPS patients, a long-term low-dose daily (once-per-cycle) treatment
regime is indicated so that each cycle is slightly lengthened to bring it
to approximately 24 hours.

Example 6

Dose Response

[0281]FIG. 10 shows a dose response curve on SCN slices. The data fit a
one-site inhibition model. The data suggest we may be achieving the
maximum delay available using this inhibitor.

[0282]FIG. 11 shows a dose response curve on 3T3 cells. The data fit a
one-site inhibition model: The data suggest we may be achieving the
maximum delay available using this inhibitor.

[0283]It should be noted that whereas Forskolin (AC agonist) had amplitude
effect, THFA (AC inhibitor) has a period effect. This is an extremely
surprising and dramatic result.

Example 7

Use of P-Site Inhibitor According to the Present Invention

[0284]FIG. 12 shows a graph of P-site vs Gs site inhibition. THFA is the
P-site inhibitor, MDL is the Gs site inhibitor. P-site inhibitors are
preferred according to the present invention.

[0285]FIG. 13 shows a graph demonstrating that SCN period is at cellular
level. This is still true at single neuron level.

[0286]We then investigated whether any mechanistic details were implied by
the change to the SCN emission waveform.

Summary

[0287]Thus we demonstrate an important clock mechanism, and show how the
system can be reliably and reproducibly perturbed by extension of the
period of the clock further than ever before. This is useful in the
treatment of jet lag, shift lag, and related circadian tai disorders as
well as hereditary conditions such as FASPS.

[0288]We shows that cAMP signaling constitutes a new level of circadian
regulation.

[0290]We show that adenylyl cyclase inhibition leads to unprecedented
period lengthening of at least 30-31 hour periods. Inhibition has no
effect on amplitude, only period.

[0291]We demonstrate the utlity of adenyl cyclase inhibitors, preferably
P-site adenylyl cyclase inhibitors, in the treatment of
disease-associated with the circadian rhythm.

[0292]Thus the importance of cAMP as an intracellular target in alteration
of circadian rhythm is demonstrated.

Example 8

Manipulation of Circadian Rhythm in Mammals

[0293]We demonstrate effects of P-site adenylyl cyclase inhibition in mice
by administration of P-site adenylyl cyclase inhibitor. In this example
the P-site adenylyl cyclase inhibitor is THFA. FIG. 14 shows the
structure of SQ 22536 (9-(Tetrahydro-2'-furyl)adenine (THFA)) which has a
Mw of 205.2. In this example the mammals are mice.

[0305]Calculations based on the premise that after one day THFA leaves the
body at the same rate it enters, infer that serum concentration reaches
about 0.6-0.9 mM within a day or so.

[0306]Effects on the period of the circadian rhythm are monitored.

Example 9

Adenylyl Cyclase Involvement

[0307]We investigated the possibility that intracellular signalling might
sustain the mammalian clock. Buffering of [Ca2+]i attenuates
SCN circadian gene expression, and cellular rhythms are both attenuated
and desynchronised in the SCN of mice lacking the VPAC2 receptor
(Vip2r.sup.-/-) for vasoactive intestinal peptide. This receptor is
positively coupled to AC and in organotypical Vip2r.sup.-/- SCN slices,
carrying a mPer1::luciferase reporter, direct activation of AC by
forskolin enhances circadian transcriptional activity (FIGS. 15a, b). If
AC-dependent signals are necessary for normal clock function, then
suppression of AC signals in wild-type SCN should attenuate circadian
gene expression and lead to cellular desynchrony. More significantly, if
AC is part of the cellular clock, then appropriate manipulation of AC
should affect circadian period: a canonical property of the oscillator.

[0308]The contribution of endogenous AC to transcriptional cycling in
wild-type SCN slices was tested by addition of MDI, 12,330A (MDL), a
potent, irreversible AC inhibitor at the Gs.sub.α site which at the
highest doses used suppressed cAMP levels to below the sensitivity of
detection (FIG. 20a). MDL caused a rapid, dose-dependent dampening of
circadian cycling, observed with both mPERIOD2-LUCIFERASE (mPER2-LUC)
fusion protein and mPer1::luc transcriptional reporter (FIG. 15c, FIG.
20b). Circadian period was not affected. The dampening was reversible
upon washout, albeit requiring time to recover from high doses,
presumably as new AC was synthesised (FIG. 15d, FIG. 20b).
Transcriptional dampening from the SCN slice might arise from loss of
individual cellular amplitude and/or cellular desynchrony.

[0309]Video imaging of cellular circadian mPER2-LUC expression using a CCD
camera revealed a very rapid effect of 2.5 μM MDL across the SCN (FIG.
15e). During prolonged (>7 days)-treatment with an intermediate dose
or MDL (1.0 μM) it became obvious that SCN cells not only lost
circadian amplitude but also became desynchronised, phenocopying the
Vip2r.sup.-/- SCN (FIG. 15f). The effect of AC-Gs.sub.α inhibition
on the clockwork was general and not restricted to the SCN. Reversible,
dose-dependent suppression of circadian output by MDL was also evident in
organotypic kidney slices from mPER-2-LUC mice and NIH 3T3 cells
transfected with a Bmal1::luc reporter (Ueda et al 2005 Nat Genet Vol 37
pp 187-92; see FIG. 20c, d). MDL had no effect on luciferase expression
from a control, non-circadian promoter transfected into NIH 3T3 cells.

[0311]Statistical analysis was performed using Prism GraphPad® and
Statview®. Periods were calculated over ≧3 days recoding For
comparative purposes, transcriptional amplitude from PMT recordings was
calculated as 100× (post-treatment emission
peak-trough/pre-treatment peak-trough). In all cases error bars indicate
SEM about the mean where n≧3.

Example 10

P-site Inhibition

[0312]Loss of SCN circadian amplitude and cellular synchrony in the
absence of functional AC does not necessarily establish a role for
AC/cAMP signalling in the core oscillator because both may arise from
defective clock output. We therefore tested the effect of an alternative
form of AC inhibition by using 9-(tetrahydro-2-furyl)-adenine (THFA), a
non-competitive AC inhibitor at the purinergic site (p-site). In contrast
to MDL, THFA did not alter basal levels of cAMP in fibroblast cultures,
but slowed the rate of cAMP synthesis and thereby attenuated peak levels
in fibroblasts (FIG. 20e). In mPer1::luc and mPER2-LUC SCN slices THFA
caused a robust and dose-dependent increase of circadian period, from ca.
24 to 31 hours (FIGS. 16a, b, c), with some dampening of transcriptional
amplitude at higher concentrations. The dose-response was consistent with
a one-site inhibition model, saturating at around 2 mM, and was rapidly
reversible upon drug washout. Moreover, CCD imaging revealed that THFA
increased period in individual neurons across the SCN (FIG. 16c).

[0313]The circadian effect of p-site inhibition was confirmed with
additional non-competitive inhibitors: 2'5'-dideoxyadenosine and
9-cyclopentyl adenine (FIG. 16d). Period was lengthened in both m
Per1:luc and PER2::LUC SCN slices. The effect of THFA on SCN period was
additive to that of the Clock mutation (FIG. 16e), suggesting THFA acts
in addition to, and independently of, E-box mediated trans-activation by
CLOCK. Importantly, 2 mM THFA also caused a dramatic increase in
circadian period in all peripheral tissues tested from mPER2-LUC mice
(FIG. 16f), whilst in fibroblasts transfected with mBmal::luc reporter
there was an even more pronounced effect, circadian period lengthening
from ca. 21 to 31 hours (FIGS. 16g, h). Inhibition of AC also lengthened
circadian period of wheel-running behaviour when delivered to the SCN of
centrally cannulated mice. Following recovery from surgery, the
activity/rest cycles of vehicle-treated mice were clearly entrained to
the light-dark cycle, activity onset coinciding with lights off, and upon
release into continuous dim red light they free-ran with a period very
close to 24 hours (FIGS. 17a, b). In contrast, in all nine mice that
received chronic central THFA, free-running circadian period was
significantly (p<0:01, t-test) lengthened upon transfer to continuous
dim red light.

Example 11

JNK Inhibition

[0314]Protein kinase A (PIA) is a candidate for mediating circadian
effects of cAMP, so mPer1::luc and mPER2-LUC SCN slices were incubated
with a series of well characterized PKA inhibitors targeted against
either the regulatory or the catalytic sub-unit. Surprisingly, there was
no significant effect on either circadian amplitude or period (FIG. 21).

[0315]Epac1/2 are reported to be alternative mediators of AC signalling.
To test their putative role mPer1:Lluc SCN slices were incubated with
Sp-8-CPT-2'-O-Me-cAMPS, a specific Epac agonist. This had no significant
effect on rhythmic transcriptional amplitude or period. When mPER2-LUC
SCN slices were treated with 2.5 μM MDL to induce dampening, however,
the Epac-specific agonist dramatically restored circadian output, higher
amplitude rhythms persisting for several cycles (FIG. 18a, FIG. 22a);
thereby showing that activation of downstream Epac activity can
compensate for the effect of AC inhibition of circadian gene expression;
CCD recording revealed that Epac agonist transiently activated and
re-synchronised circadian gene expression in MDL-treated SCN neurons
(FIG. 18b). Further, when fibroblasts carrying the Bmal1::luc reporter
were transfected with a known inhibitor of Epac, HA-Rap1(S17N), circadian
gene expression was markedly dampened (FIG. 18c, FIG. 22b). Wild-type
Rap1, which is not effective at Epac had no effect. Furthermore, RTAi
knock-down of endogenous Epac1 and Epac 2 in fibroblasts similarly
dampened circadian gene expression (FIG. 18d, FIG. 22c). Epac is reported
to activate c-Jun N-terminal kinase (JNK) p46, and pJNK in turn activates
gene expression through transcription factors of the AP-1 family. Given
that Period genes contain AP-1 regulatory sequences, we conceived a model
whereby cyclical activation of cAMP/Epac/JNK/AP-1 drives circadian
pacemaking. Consistent with this model, Epac agonist increased pJNK (but
not pCREB) fibroblasts given 5 μM MDL (FIGS. 22d, e), and JNK
inhibition lengthened circadian period in SCN slices and fibroblasts
comparable to the effect of AC inhibition (FIGS. 18e, f, FIGS. 22f, g).
Importantly, the effect of JNK inhibition was additive to that of THFA,
leading to unprecedented lengthening of the SCN and fibroblast clockwork
to around 36 hours. In contrast, IBMX which lengthened period in
fibroblasts did not enhance the effect of THFA, presumably because both
act via perturbing cAMP levels and so have redundant effects (FIG. 22g).

Summary

[0316]Our results demonstrate that AC activity, likely mediated via
Epac/JNK, determines the amplitude and period of circadian
transcriptional loops, and that interference with AC signalling
desynchronizes SCN clock cells, probably because loss of circadian
amplitude compromises inter-neuronal signals, such as VIP release, that
constitute an output from the core oscillator. We therefore propose that
circadian timing in mammals is sustained and its period determined by a
reciprocal interplay in which transcriptional/post-translational feedback
loops and neural activity drive intracellular rhythms of AC signalling.
This cytosolic-rhythm in turn potentiates transcriptional cycles i.e.
clock output constitutes an input into the current and/or subsequent
cycle (FIG. 19). The dependence of transcriptional cycles on cAMP
signalling explains the compromised circadian gene expression and
behaviour in Vip2r.sup.-/- mice, and is likely applicable to local
clockworks in major organs where afferent signals other than VIP regulate
AC activity. It may also extend to the Drosophila clock, which is
maintained by the PDF receptor (a homologue of VPAC2 receptor) that
activates AC. The differential effect of AC inhibitors (Gsα
vs. p-site) likely reflects their particular actions on AC kinetics, and
non-competitive p-site inhibitors such as THFA may be of broad
therapeutic application where either acute (jet-lag, shiftwork) or
maintained (Familial Advanced Sleep Phase Syndrome) extension of
circadian period in all body tissues is indicated.

[0317]All publications mentioned in the above specification are herein
incorporated by reference. Various modifications and variations of the
described methods and system of the present invention will be apparent to
those skilled in the art without departing from the scope of the present
invention. Although the present invention has been described in
connection with specific preferred embodiments, it should be understood
that the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the described
modes for carrying out the invention which are obvious to those skilled
in biochemistry and biotechnology or related fields are intended to be
within the scope of the following claims.